• Reproductive and Developmental Biology
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• v.30 no.4
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• pp.229-234
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• 2006

We have generated transgenic mice that expressed mouse extracellular superoxide dismutase (EC-SOD) in their skin. In particular, the expression plasmid DNA containing human keratin K14 promoter was used to direct the keratinocyte-specific transcription of the transgene. To compare intron-dependent and intron-independent gene expression, we constructed two vectors. The vector B, which contains the rabbit -globin intron 2, was not effective for mouse EC-SOD overexpression. The EC-SOD transcript was detected in the skin, as determined by Northern blot analysis. Furthermore, EC-SOD protein was detected in the skin tissue, as demonstrated by Western blot analysis. To evaluate the expression levels of EC-SOD in various tissues, we purified EC-SOD from the skin, lungs, brain, kidneys, livers, and spleen of transgenic mice and measured its activities. EC-SOD activities in the transgenic mice skin were approximately 7 fold higher than in wild-type mice. These results suggest that the mouse overexpressing vector not only induces keratinocyte-specific expression of EC-SOD, but also expresses successfully functional EC-SOD. Thus, these transgenic mice appeared to be useful for the expression of the EC-SOD gene and subsequent analysis of various skin changes, such as erythema, inflamation, photoaging, and skin tumors.

• BMB Reports
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• v.37 no.2
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• pp.139-143
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• 2004

The antioxidant responsive element (ARE) is a cis-acting regulatory element of genes encoding phase II detoxification enzymes and antioxidant proteins, such as NAD(P)H: quinone oxidoreductase 1, glutathione S-transferases, and glutamate-cysteine ligase. Interestingly, it has been reported that Nrf2 (NF-E2-related factor 2) regulates a wide array of ARE-driven genes in various cell types. Nrf2 is a basic leucine zipper transcription factor, which was originally identified as a binding protein of locus control region of ss-globin gene. The DNA binding sequence of Nrf2 and ARE sequence are very similar, and many studies demonstrated that Nrf2 binds to the ARE sites leading to up-regulation of downstream genes. The function of Nrf2 and its downstream target genes suggests that the Nrf2-ARE pathway is important in the cellular antioxidant defense system. In support of this, many studies showed a critical role of Nrf2 in cellular protection and anti-carcinogenicity, implying that the Nrf2-ARE pathway may serve as a therapeutic target for neurodegenerative diseases and cancers, in which oxidative stress is closely implicated.

• Journal of Integrative Natural Science
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• v.6 no.4
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• pp.193-196
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• 2013

Haemoglobin is an interesting physiologically significant protein composed of specific functional prosthetic haem and globin moieties. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins (Hbs). Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on Amaurornis phoenicurus Hb. Crystals were grown by the hanging drop vapor-diffusion method using PEG 2000 and NaCl as precipitants. The crystals belonged to the primitive monoclinic system $P2_1$, with unit-cell parameters $a=65.33{\AA}$, $b=93.14{\AA}$, $c=98.54{\AA}$, ${\beta}=100.48^{\circ}$; a complete data set was collected to a resolution of $2.6{\AA}$. The Matthews coefficient of $2.30{\AA}^3Da^{-1}$ for the crystal indicated the presence of two ${\alpha}_2{\beta}_2$ tetramers in the asymmetric unit.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.4
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• pp.2355-2359
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• 2013

Vulva cancer is rare among all gynecological cancer worldwide, including Thailand, and mainly affects older women. Persistent high risk type infection of human papillomavirus (HPV) is the one important factor for developing cancer. In this study, we focused on HPV DNA investigation and type-specific distribution of HPV in 25 formalin-fixed paraffin-embedded (FFPE) samples collected from Thai women with vulva cancer histologically confirmed by the National Cancer Institute, Thailand, during 2003-2011. HPV DNA detection and genotyping were undertaken with polymerase-chain reaction and enzyme-immunoassay using GP5+/bio6+ consensus specific primers and digoxigenin-labeled specific oligoprobes, respectively. Human ${\beta}$-globin genes was used as the internal control. Our results showed that 44% (11/25) of all vulva cancer samples were HPV-positive. All of them are high risk HPV type infection, detected as single (63.64%, 7/11) and/or double infections (4/11, 36.36%). HPV 16 was the most common type identified in vulva cancer, followed by HPV 35, 33, 18 and 58. In conclusion, this study presented that HPV-16 is observed at the highest frequency in this cancer, similar to cervical cancer, with HPV 18 being less frequent. Although the sample size was small and could not represent overall incidence and prevalence in Thai women, these preliminary data for vulva cancer are of interest since they reinforce the necessity for HPV screening or vaccination in Thailand.

• Journal of Environmental Science International
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• v.20 no.10
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• pp.1221-1232
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• 2011

We investigated the toxic effects of difenoconazole on the development in the African clawed frog, Xenopus laevis. To test the toxic effects, frog embryo teratogenesis assays using Xenopus were performed. Embryos were exposed to various concentrations of difenoconazole (0-30 ${\mu}M$). $LC_{100}$ for difenoconazole was 30 ${\mu}M$, and the $LC_{50}$ determined by probit analysis was 27.19 ${\mu}M$. Exposure to difenoconazole concentrations ${\geq}$5 ${\mu}M$ resulted in 10 different types of severe external malformation. Histological examinations revealed dysplasia of the eye, heart, liver, somatic muscle, and swelling of the pronephric ducts. The tissue-specific toxic effects were investigated with an animal cap assay. Blood cells were normally induced at a high frequency by mSCF and activin A. However, the induction of blood cells was strongly inhibited by the addition of difenoconazole. Electron micrographs of tested embryos showed the degeneration of somatic muscle and the shrinkage of microvilli on pronephric duct. The gene expression of cultivated animal cap explants was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR). It revealed that the expression of the blood-specific marker(${\beta}$-globin II) and muscle-specific marker (XMA) were more strongly inhibited than the neural-specific marker(XEn2) by the addition of difenoconazole.

• Journal of Environmental Science International
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• v.19 no.8
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• pp.1001-1012
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• 2010

We investigated the toxic effects of tebuconazole on development in the African clawed frog, Xenopus laevis. To test the toxic effects, frog embryo teratogenesis assays using Xenopus were performed. Embryos were exposed to various concentrations of tebuconazole($0-100\;{\mu}M$). $LC_{100}$ for tebuconazole was $100\;{\mu}M$, and the $LC_{50}$ determined by probit analysis was $82.35\;{\mu}M$. The exposure to tebuconazole concentrations ${\geq}40\;{\mu}M$ resulted in 11 different types of severe external malformations including gut dysplasia. Histological examinations revealed various dysplasia in the eye, heart, liver, intestine, somatic muscle, and in the pronephric ducts. The tissue-specific toxic effects were investigated with an animal cap assay. Blood cells are generally induced at a high frequency by the combination of mSCF and activin A, however, the induction of blood cells was strongly inhibited by the addition of tebuconazole. Electron micrographs of tested embryos showed many of multivesicular bodies and dysplasia of photo-receptive cell, however, the somatic muscle degeneration was not severe. The gene expression of cultivated animal cap explants was investigated by reverse transcriptase-polymerase chain reaction and revealed that expression of the blood-specific marker, $\beta$ globin II and muscle-specific marker, muscle actin were more strongly inhibited than the neural-specific marker, XEn2.

• Journal of the Korean Magnetic Resonance Society
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• v.18 no.1
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• pp.30-35
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• 2014

The transcription factor CP2 regulates various biological systems at diverse tissues and cells. However, none of the four CP2 isoforms has been solved in structure yet. In particular, two different regions of the CP2b isoform have been characterized to interact with the PIAS1 in nucleus to regulate the ${\alpha}$-globin gene expression. Among them, in this study, the region encompassing residues 251-309 of CP2b was prepared as a recombinant protein and its solution structure was characterized by NMR spectroscopy. The results indicated that the CP2b(251-309) fold belongs to typical IDRs (intrinsically disordered regions), likely to facilitate promiscuous interactions with various target proteins. Unfortunately, however, its interaction with the N-terminal domain of PIAS1 (residues 1-70), which has been identified as one of the CP2b-binding sites, was not observed in the NMR-based titration experiments. Therefore, it could be postulated that the 251-309 region of CP2b would not contact with the PIAS1(1-70), but alternatively interact with another CP2b-binding region that encompasses residues 400-651 of PIAS1.

• BMB Reports
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• v.31 no.4
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• pp.413-417
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• 1998

Transgenic mice containing a bovine ${\beta}-Casein/Human$ lysozyme fusion gene (pBZ) were generated in order to produce human lysozyme in their milk. The expression vector was a quadripartite fusion consisting of a 2 kb upstream DNA of the bovine ${\beta}-casein$ gene, human lysozyme gene, intron II of the rabbit ${\beta}-globin$ gene, and the polyadenylation/termination signals of SV40 DNA. Fertilized mouse zygotes were microinjected with pBZ, then transferred into the oviduct of foster mothers. Out of 20 mice born, 11 survived until postweaning and three were identified as positivetransgenic by Southern blot analysis (one male and two females). The founder mice were mated to BCFl mice to produce transgenic progeny. It was confirmed by RT-PCR and Northern blot analyses that the transgene was specifically expressed in the mammary gland of the founder mice. Furthermore, the artificial introns within the transgenic RNA was proven to be correctly spliced out as judged by RT-PCR analysis. These results indicated that transgenic mice generated in this study properly expressed the human lysozyme RNA in their mammary gland.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.2
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• pp.579-583
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• 2015

This study was conducted on female patients with different gynecological problems attending the gynecology out-patient departments of two tertiary care hospitals in Peshawar city of Khyber Pakhtunkhwa, Pakistan between August 2012 and October 2013. The 200 patients had an age range of 21-65 years. Smears were taken with cervical brushes and preserved in preservative medium and processed for manual liquid based cytology (MLBC) for Pap staining. Out of 200 collected samples, 30 samples were found inadequate on cytology. Of the remaining 170 samples, 164 (96.47%) were normal, 5 (2.94%) were of atypical squamous cells of unknown significance (ASCUS) and 1 (0.6%) was of high grade squamous intraepithelial lesion (HSIL). On PCR all the samples were positive for beta globin gene fragment including those reported inadequate on cytology. Out of the 5 ASCUS samples, 2 samples were positive for HPV, one each for HPV 16 and HPV 18, and the rest of the 3 samples were negative for HPV DNA. The 1 sample of HSIL was positive for HPV 16 on PCR. Out of 164 normal samples on cytology, only 1 sample was HPV 16 positive. So overall, 4 (2%) out of 200 samples were positive for HPV DNA, where 3 were HPV 16 (1.5%), and 1 was HPV 18 (0.5%) positive, and thus the ratio of infection with of HPV 16 to HPV 18 was 3:1 in the general population. In conclusion, PCR based HPV detection is a more sensitive method for screening of HPV infection than cytology as sample inadequacy does not affect the results. However, it can be combined with cytology methods in a HPV positive female to achieve the maximum results.

• BMB Reports
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• v.28 no.1
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• pp.57-61
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• 1995

The expression of a recombinant human lactoferrin is reported in mouse HC11 mammary epithelial cells. Expression of human lactoferrin (hLF) was achieved by placing its cDNA under the control of the bovine ${\beta}$-casein gene. To improve the hLF expression level in a cell culture system, two artificial introns were also introduced to construct expression vectors. One intron was a hybrid-splice signal consisting of bovine ${\beta}$-casein intron 1 and rabbit ${\beta}$-globin intron II. The other intron was a DNA fragment spanning intron 8 of the bovine ${\beta}$-casein gene. The hybrid intron moderately elevated hLF expression, whereas intron 8 alone did not express any detectable amount of hLF as judged by Northem and Western blot analyses. When the two introns were used together they contributed to a synergistic elevation of hLF expression. These data indicate that artificial introns on both sides of the hLF cDNA were necessary to increase expression of cDNA.